适配体介导主动靶向与胞内触发释药的多功能纳米递药系统的构建及抗前列腺癌研究

Geldanamycin and some of its derivatives (such as 17-AAG) exhibited potent anti-cancer activity as the excellent Hsp90 inhibitors, but the limitations of liver toxicity, metabolical unstablity definitely hinder their clinical development. In the previous work, the derivative G2111 was synthesized and found to have excellent anti-prostate cancer acivity with low hepatotoxicity, and it is likely to become a new target candidate drug for the treatment of prostate cancer. However, the poor solubility of G2111 is the main hindrance for its further development. In the present study, a novel intelligent and targeted nano-delivery system for G2111 is designed, by utilizing PLGA which contains disulfide bonds as the core, the lecithin and DSPE-PEG as the shell, and the aptamer A10 which could specially recognize prostate-specific membrane antigen as the “target head”. The novel drug delivery system possesses many advantages in the treatment of prostate cancer. Firstly, the system has the long circulating action and good stability owing to the PEG shell. Secondly, the system can control drug release via the disulfide bond in the PLGA core. Thirdly, it is the aptamer A10-oriented role in identifying prostate cancer cells. The construction of nano drug delivery system, characeristics, in vitro and in vivo antitumor activities will be investigated systematically in this work. Hence, this project could provide suggestions and theoretical basis for the clinical transformation of G2111, and will open up a new path for the treatment of prostate cancer.

格尔德霉素（GA）及其衍生物17-AAG等是著名的Hsp90抑制剂，由于肝毒性、代谢不稳定等局限，尚未能应用于临床。前期研究我们对GA进行结构修饰，通过筛选获得了衍生物G2111，G2111不仅在抗前列腺癌方面展现出了独特优势而且其肝毒性较低，极有可能成为新型靶向治疗前列腺癌的候选药物。然而，G2111与17-AAG类似，水溶性较差，给G2111的开发研究造成阻力。本课题拟构建一种以含有二硫键的PLGA聚合物为载药内核，以卵磷脂和DSPE-PEG为仿生型外壳，以可选择性识别前列腺特异性膜抗原的适配体A10为“靶头”的新型靶向递药系统。该递药系统可能具有多种功能，既可以在PEG层的保护下进行长循环、保持稳定性，也可以发挥适配体的导向基团识别作用，实现前列腺癌的靶向治疗，同时可以在肿瘤细胞内实现触发释药。通过对本课题的研究拟为G2111的临床转化研究提供基础，同时为前列腺癌的化疗提供新途径。

本项目结合聚合物纳米粒和脂质体作为药物载体的优势，设计建立了一种以含有二硫键的PLGA聚合物为载药内核，以卵磷脂和DSPE-PEG为仿生型外壳的复合型纳米粒，并通过PEG 末端的功能基团在纳米粒表面同时修饰可选择性识别前列腺癌细胞膜表面表达的PSMA的适配体A10。所构建的载G2111靶向纳米粒（G2111-A10-PEG-PLGA）平均粒径为128 nM，载药量为5.22%，GSH响应试验证明所构建的G2111-A10-PEG-PLGA纳米粒对GSH表现出了良好的响应性，进而预示含有二硫键的聚合物内核在细胞外可以保持稳定性，而一旦进入细胞，在GHS的作用下二硫键断裂，聚合物内核结构破坏，所载药物被迅速释放出来，从而实现药物在细胞内触发式释放，利于药物尽快起效。纳米粒生物相容性评价结果显示，空白载体对红细胞及前列腺癌细胞LNCaP展示出了良好的生物相容性，且G2111被A10-PEG-PLGA纳米粒装载后依旧表现出对肝细胞良好的相容性。细胞学评价方面显示，该复合型纳米粒表面经PSMA适配体A10修饰后可以显著提高纳米粒被细胞摄取的能力，增强纳米粒的靶向性。与G2111溶液组相比较，所有纳米粒制剂体外抗肿瘤活性均显著高于溶液组；靶向纳米粒对LNCaP细胞毒作用均比普通纳米制剂高，IC50值显著降低（P < 0.05），分别为117.9±10.5 nM（G2111-A10-PEG-PLGA）、143.9±14.1 nM（G2111-PEG-PLGA）及177.6±16.7 nM（G2111-Sol）。由此可见，所构建的G2111-A10-PEG-PLGA纳米粒，不仅可以很好的弥补G2111理化性质的缺陷（水溶性差），更重要的是通过A10的修饰及纳米制剂自身的优点，可显著提高G2111的体外抗肿瘤活性。大鼠体内药动学结果显示，所构建的纳米粒由于PEG化的磷脂壳层可在纳米粒表面形成一层PEG水化层，进而增加了纳米粒表面的亲水性，降低纳米粒在血液循环中与调理素相结合，从而延长纳米粒在体内的循环时间，提高其体内药物的稳定性。基于本项目的研究结果，将为G2111的继续研发及临床转化提供有效的实验数据支持，同时为前列腺癌的化疗提供新方向。